Hyaluronidase from the Hirudinaria manillensis isolation, purification and recombinant method of production

ABSTRACT

The present invention relates to the isolation, purification and characterization of a hyaluronidase which derives from the tropical leech  Hirudinaria manillensis . Therefore, according to this invention, the enzyme was called “manillase”. The invention is furthermore concerned with the recombinant method of production of manillase which includes the disclosure of DNA and amino acid sequences as well as of expression vectors and host systems. Finally, the invention relates to the use of manillase for therapeutic purposes, for example, for the treatment of myocardial diseases, thrombotic events and tumors.

The present invention relates to the isolation, purification andcharacterization of a novel hyaluronidase which derives from thetropical leech Hirudinaria manillensis. Therefore, according to thisinvention the new enzyme is called “manillase”. The invention isfurthermore concerned with the recombinant method of production ofmanillase which includes the disclosure of DNA and amino acid sequencesas well as of expression vectors and host systems. Finally, theinvention relates to the use of manillase for therapeutic purposes, forexample, for the treatment of myocardial diseases, thrombotic events andtumors.

Hyaluronic acid or hyaluronan (HA) is a linear unbranched highmolecular-weight (2–6×10⁶) glycosaminoglycan, composed of a repeatingdisaccharide structure GlcNAc(β1–4)GlcUA. Its carboxyl groups are fullyionized in the prevailing pH of extracellular fluids, whether normal orpathological. HA belongs together with the chondroitin sulphates,keratan sulfates and heparins to the group of glycosaminoglycans(Jeanloz R. W., Arthr Rheum., 1960, 3, 233–237). In contrast with otherunmodified glycosaminoglycans (GAG), it has no sulfate substitution orcovalently linked peptide, and its chain length and molecular weight areusually very much greater. HA is ubiquitously distributed in connectivetissues and has been found in virtually all parts of the body afterintroduction of improved fixation method (Hellström S. et al., 1990,Histochem. J., 22, 677–682) and the specific histochemical method withthe use of hyaluronan-binding peptides (HABP). It is present duringdevelopment and maturity in tissues of neuroectodermal origin as well.

The term hyaluronidase refers generally and according to this inventionto an enzyme, which acts on hyaluronic acid, irrespective of activitytowards other substrates.

Hyaluronidase was first isolated from microorganisms and later frommammalian testis which is now its main source (Meyer K. in The Enzyme,1971, 307).

According to the reaction mechanism, hyaluronidases were divided intothree main groups.

In the first group microbial enzymes are combined that act on theirsubstrates by β-elimination producing Δ4,5-unsaturated disaccharides.The enzyme must therefore be named hyaluronate lyases, EC 4.2.99.1.

The second group, hyaluronoglucosamimidase or testicular-typehyaluronidase (EC 3.2.1.35) acts as an endo-N-acetyl-β-D-hexosaminidasedegrading HA to smaller fragments, in the first place tetrasaccharidewith the hexosamine moiety at the free reducing end. Enzymes withsimilar properties to the testis hyaluronidase have been obtained fromtadpoles, snake venom, bee venom, numerous animal tissues, human serumand other sources. It is well know that hyaluronidase from testis hasalso transglycosylase activity (Weissman B. et al., J. Biol. Chem.,1954, 208, 417–429). The enzymes belonging to this group ofhyaluronidases exhibit enzymatic activity not only towards hyaluronatebut also towards chondroitin-4-sulfate, chondroitin-6-sulfate,chondroitin and dermatan sulfate.

The third group consists of hyaluronoglucuronidase (EC 3.2.1.36), whichacts as an endo-β-glucuronidase. This enzyme was isolated from theHirudo medicinalis leeches (Yuki H. & Fishman W. H.; J. Biol. Chem.1963, 238, 1877–79) and is absolutely specific for HA. Chondroitinsulfate, dermatan and heparin are not substrates for this hyaluronidase.It degrades only hyaluronic acid to tetrasaccharide with the glucuronicacid at the free reducing end (Linker A. et al., J. Biol. Chem., 1960,235, 924–27). Opposite to mamalian endo-βglucosamimidases, heparin hasno influence on the activity of this leech hyaluronidase. Therefore, itcan be coadministered to a patient together with a heparin and itsderivatives extensively used as anticoagulants. A hyaluronic acidspecific endo-beta-glucuronidase (called “Orgelase”) from species(Poecilobdella granulosa) of the sub-family Hirudinariinae (includingthe genera Hirudinaria, Illebdella, Poecilodbella, Sanguisoga) ofbuffalo leeches was disclosed in EP 0193 330 having a molecular weightof about 28.5. Hyaluronidases have many practical in vivo and in vitroapplications. Intravenous administration of hyaluronidase has beenproposed for treatment of myocardial infraction (Kloner R. A et al.,Circulation, 1978, 58, 220–226; Wolf R. A. et al., Am. J. Cardiol, 1984,53, 941–944; Taira A. et al., Angiology, 1990, 41, 1029–1036).Myocardial infraction represents a common form of non-mechanical injury;namely severe cell damage and death, caused in this instance by suddencellular hypoxia. In an experimental myocardial infraction induced inrats (Waldenström A. et al., 1991, J. Clin. Invest., 88, 1622–1628), HAcontent of the injured (infracted area) heart muscle increased within 24h to reach nearly three times normal after 3 days, and was accompaniedby interstitial oedema. The relative water content of infracted areasalso increased progressively reaching a maximum value by day 3 and wasstrongly correlated with the HA accumulation. The same association ofincreased HA content with oedema has been observed in experimental heartand renal transplant rejection (Hällgren R. et al., J. Clin. Invest.,1990, 85, 668–673; Hällgren R. et al., J. Exp. Med., 1990, 171,2063–2076) in rejection of human renal transplants (Wells A. et al.Transplantation, 1990, 50, 240–243), lung diseases (Bjermer A. et al.,Brit. Med. J., 1987, 295, 801–806) and in idiopathic interstitialfibrosis (Bjermer A. et al., Thorax, 1989, 44, 126–131). All thesestudies provide not only evidence of increased HA in acute inflammation,but demonstrate its part in the local retention of fluid mainlyresponsible for the tissue swelling and influencing both the mechanicaland electrophysiological functions of heart.

These results can explain the mechanism of the action of hyaluronidasesused in clinical trials. It was reported that hyaluronidase treatmentlimited cellular damage during myocardial ischemia in rats, dogs and man(Maclean D. et al. Science, 1976, 194, 199). The degradation of the HAcan be followed by the reduction of tissue water accumulation, reductionof the tissue pressure and finally better perfusion.

It has been shown that hyaluronidases as well as hyaluronidasecontaining extracts from leeches can be used for other therapeuticpurposes. Thus, hyase therapy, alone or combined with cyclosporine,resulted in prolonged graft survival (Johnsson C. et al. TransplantInter. in press). Hyases (“spreading factor”) in the broadest sense areused to increase the permeability of tissues for enhancing the diffusionof other pharmacological agents (e.g. in combination with cytostatics inthe treatment of cancer tumors). Furthermore, it could be demonstratedthat hyaluronidases are useful in tumor therapy acting as angiogenesisinhibitor and as an aid to local drug delivery in the treatment oftumors, for the treatment of glaucoma and other eye disorders and asadjunct to other therapeutic agents such as local anaesthetics andantibiotics. A general overview of the therapeutic use and relevance isgiven in the review article of Farr et al. (1997, wiener MedizinischeWochenschrift, 15, p. 347) and literature cited therein. Therefore,there is a need for an active compound such as hyaluronidase. However,the known and available hyaluronidases are either not stable(hyaluronidase from Hirudo medicinalis, Linker et. al., 1960, J. Biol.Chem. 235, p. 924; Yuki and Fishman, 1963, J. Biol. Chem. 238, p. 1877)or they show a rather low specific activity (EP 0193 330, Budds et al.,1987, Comp. Biochem. Physiol., 87B, 3, p. 497). Moreover, none of theknown hyaluronidases are available in recombinant form which is anessential prerequisite for intensive commercial use.

This invention discloses now for the first time a new hyaluronidasewhich was isolated and purified from Hirudonaria mannilensis as well asa recombinant version of said enzyme obtained by bioengineeringtechniques.

Thus, it is an object of this invention to provide a purified proteinisolated from the leech species Hirudinaria manillensis having thebiological activity of a hyaluronidase which is not influenced in itsacvtivity by heparin and characterized in that it has a molecular weightof 53–60 kD dependent on glycosylation. The new protein, which is called“manillase”, is glycosylated in its native form having a molecularweight of ca. 58 kD (±2 kD) and four glycoforms. However, thenon-glycosylated protein is object of the invention as well, obtainableby enzymatic or chemical cleavage of the sugar residues according tostandard techniques. The non-glycosylated enzyme of the invention has amolecular weight of about 54 (±2) as measured by SDS-PAGE.

Direct comparison shows that the hyaluronidase disclosed in EP 0193 330(“orgelase”) has under the same conditions a molecular weight of about28 and contains a lot of impurities such as hemoglobin.

Native manillase according to this invention has a pH optimum of6.0–7.0, an isoelectric point of 7.2–8.0 and has the amino acid sequencedepicted in FIG. 7.

Surprisingly manillase obtained by a preparative purification procedure(see below) has an extremely high specific activity of 100–150,preferably of 110–140 (WHO) kU/mg protein whereas the specific activityof orgelase is about 1,2 kU/mg only. Moreover, orgelase has a lower pHoptimum (5.2–6.0) as compared with manillase. Manillase is notinfluenced, like orgelase, by heparin.

Furthermore it is an object of the invention to provide a process forisolating and purifying manillase comprising the following steps

-   (i) homogenization of heads of leeches of the species Hirudinaria    manillensis with an acid buffer and centrifugation,-   (ii) ammonium sulfate precipitation of the supernatant of step (i),-   (iii) cation exchange chromatography,-   (iv) concanavalin A affinity chromatography-   (v) hydrophobic interaction chromatography-   (vi) affinity chromatography on matrices coated with hyaluronic acid    fragments-   (vii) gel permeation chromatography, and optionally-   (viii) enzymatic or chemical deglycosylation of the purified    protein.

The process steps disclosed above guarantee that the protein accordingto the invention can be obtained with such a high biological enzymeactivity. Therefore, it is a further object of this invention to providea protein having the biological activity of a hyaluronidase which is notinfluenced in its activity by heparin and having a molecular weight of53–60 dependent on glycosylation which is obtainable by the processsteps indicated above and in the claims and which has preferably aspecific enzyme activity of >100 kU/mg protein. The term “unit” relatesbelow and above to “international units” (IU).

The invention discloses a process of making recombinant manillase whichincludes respective DNA molecules, vectors and transformed host cells.

Therefore, it is an object of this invention to provide a DNA sequencecoding for a protein having the properties of native manillase.

It could be also shown, that at least three further clones with slightlydifferent DNA sequences could be selected which are coding for proteinswith manillase (hyaluronidase) properties having slightly differentamino acid sequences.

The specified clones have the DNA sequences depicted in FIGS. 8, 9 and10 (upper sequence) which are an object of this invention too as well asexpression vectors containing said sequences and host cells which weretransformed with said vectors.

In addition, it is object of this invention to provide a recombinantprotein with the biological activity of a hyaluronidase and a molecularweight of 55–59 kD dependent on glycosylation having any amino acidsequence depicted in FIGS. 8, 9 and 10 (lower sequence) or a sequencewhich has a homology to said sequences of at least 80%. The term“manillase” includes all these proteins having the above-specifiedproperties.

The native as well as the recombinant protein(s) may be used as amedicament which can be applied to patients directly or withinpharmaceutical compositions. Thus, it is a further aspect of thisinvention to provide a recombinant or native protein as defined aboveand below applicable as a medicament and a respective pharmaceuticalcomposition comprising said protein and a pharmaceutically acceptablediluent, carrier or excipient therefor.

The pharmaceutical compositions of the invention may containadditionally further active pharmaceutical compounds of a highdiversity. Preferred agents are anticoagulants which do not inhibit orinfluence the biological and pharmacological activity of the proteinaccording to the invention. Such anticoagulants can be, for example,heparin, hirudin or dicoumarin, preferably, heparin. Thus, it is anobject of the present invention to provide a pharmaceutical compositioncomprising additionally a pharmacologically active compound, preferablyheparin.

In connection with use in human or veterinary therapy the proteinaccording to this invention acts preferably as dispersal agent(“spreading” factor) or supports penetration through tissue and skin.Thus, manillase can be used as an adjunct of other substances (such asan local anaesthetic) e.g. in the field of chemotherapy of tumors, fortreatment of disorders and diseases with respect to acute myocardialischemia or infarction, for treatment of glaucoma and other eyedisorders, e.g. to improve the circulation of physiological fluids inthe eye, for treatment of skin and tissue grafts to remove congestionand improve circulation, as drug delivery system through the skin,membranes, other tissue, as an agent to remove the hyaluronic acidcapsule surrounding certain pathogenic microorganisms or certain tumorsand cancerous tissues, and as an inhibitor of angiogenesis which can beused as anti-thrombotic and anti-tumor agent.

Therefore, the use of manillase as defined above and below in themanufacture of a medicament for treating especially myocardial,cardiovascular and thrombotic disorders and tumors is an object of thisinvention.

As used herein, the term “pharmaceutically acceptable carrier” means aninert, non toxic solid or liquid filler, diluent or encapsulatingmaterial, not reacting adversely with the active compound or with thepatient. Suitable, preferably liquid carriers are well known in the artsuch as sterile water, saline, aqueous dextrose, sugar solutions,ethanol, glycols and oils, including those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil andmineral oil.

The formulations according to the invention may be administered as unitdoses containing conventional non-toxic pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles which are typical forparenteral administration.

The term “parenteral” includes herein subcutaneous, intravenous,intra-articular and intratracheal injection and infusion techniques.Also other administrations such as oral administration and topicalapplication are suitable. Parenteral compositions and combinations aremost preferably adminstered intravenously either in a bolus form or as aconstant fusion according to known procedures. Tablets and capsules fororal administration contain conventional excipients such as bindingagents, fillers, diluents, tableting agents, lubricants, disintegrants,and wetting agents. The tablets may be coated according to methods wellknown in the art.

Oral liquid preparations may be in the form of aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives like suspending agents, emulsifying agents,non-aqueous vehicles and preservatives.

Topical applications may be in the form of aqueous or oily suspensions,solutions, emulsions, jellies or preferably emulsion ointments.

Unit doses according to the invention may contain daily required amountsof the protein according to the invention, or sub-multiples thereof tomake up the desired dose. The optimum therapeutically acceptable dosageand dose rate for a given patient (mammals, including humans) depends ona variety of factors, such as the activity of the specific activematerial employed, the age, body weight, general health, sex, diet, timeand route of administration, rate of clearance, enzyme activity(units/mg protein), the object of the treatment, i.e., therapy orprophylaxis and the nature of the disease to be treated.

Therefore, in compositions and combinations such as with anticoagulantslike heparin in a treated patient (in vivo) a pharmaceutical effectivedaily dose of the protein of this invention (manillase) is between about0.01 and 100 mg/kg body weight (based on a specific activity of 100kU/mg), preferably between 0.1 and 10 mg/kg body weight. According tothe application form one single dose may contain between 0.5 and 10 mgof manillase.

The concentration of e.g. heparin when administered together withmanillase is typically 500–4000 U (IU) over one day, however, may beincreased or diminished if necessary.

The purification of manillase of the invention was achieved as describedin detail in the examples. Table 1 depicts a preparative purificationscheme of manillase. Table 2 shows the process of enrichment of theprotein according to the invention and Table 3 indicates the comparisonof manillase with known leech hyaluronidases.

An enzyme, named manillase, cleaving hayaluronic acid has been isolatedfrom the heads of Hirudinaria manillensis leeches and purified tohomogeneity. This hyaluronidase was purified using acid-extraction,ammoniumsulfate precipitation, followed by successive chromatography oncation exchanger, Concanavalin A-Sepharose, Propyl-Fractogel, Hyaluronanfragments-Sepharose and Diol-LiChrospher columns. The hyaluronanfragments were prepared by the cleavage of the native hyaluronan withthe aid of bovine testes hyaluronidase. After purification andcharacterization of the fragments, the affinity matrices were preparedas indicated below. Such affinity matrices were applied for the firsttime for purification of the hyaluronidase. This high-performancechromatography is a technique for fast and efficient purification ofhyaluronan binding proteins. The recovery of enzyme activity after eachstep of purification was reasonably high. The results of the threeindependent preparative purifications were comparable. They resulted inhighly active samples possessing between 20 to 160 kU/mg dependent onthe degree of purification. In comparison experiments knownhyaluronidases were isolated as indicated in the prior art and theirproperties were compared with the protein according to this invention(Tab. 3).

The hyaluronidase purified according to the scheme of Tab. 1 differsfrom other leech hyaluronidases described by other authors. A similarmolecular weight was obtained under non-dissociating conditions (any βmercaptoethanol), indicating that manillase is a single subunit enzymein common with a wide range of hyaluronidase preparations from mammaliansources. This final preparation is a single subunit enzyme (FIG. 1) ofapparent molecular weight 58±2 determined with the aid of MALDI, withisoelectric point of 7.2 to 8.0.

TABLE 1 Preparative purification of manillase

TABLE 2 Purification of manillase (enrichement) from 1 kg of leech headsTotal Total ac- % Specific Puri- protein tivity re- activity ficationStep of purification Mg kU covery U/mg (fold) Stage I 31700 633.3 100 201 supernatant after extraction and acid precipitation Stage II 9530443.3 70 45 2.25 supernatant after 36% ammonium sulfate precipitationCation exchange 426.7 332.5 52.5 770 38.5 chromatography Con A affinity-41.0 166.2 26.2 4.000 200 chromatography Propyl-Fractogel 11.9 133.021.0 11000 550 chromatography Hyaluronic acid 1.9 66.4 10.5 35000 1750fragments- Sepharose affinity chromatography Diol-LiChrospher 0.307 33.25.2 108000 5400

TABLE 3 Comparison of manillase with known leech hyaluronidasesHyaluronidase Hyaluronidase “Orgelase” “Manillase” H. medicinalis H.medicinalis P. granulosa Hirudinaria manillens. comparison Linker etal.; EP 0 193 330 Invention experiment (J.Biol.Chem, 1960) Budds et al.specific activity 140 000 ~20 000 ≦100 ≦100 WHO (IU) semipurifiedunits/mg homogeneity 1 protein Mixture of no results available mixtureof SDS-PAGE homogenous proteins many proteins MALDI 4 glycoforms mainimpurity: hemoglobin molecular 58,3 kD ± 2 kD n. d. not reported 28,5 ±3 kD weight amino acid determined n. d. not reported not determinedsequence pH optimum 6.0–7.0 6.0–7.0 not reported 5,2–6.0 pI 7.5–8,0 n.d. n. d. n. d. hydrophobicity binding to Propyl- no binding to HIC at 2MPropyl-HIC at ammonium sulfate 2M ammonium sulfate activity no influencenot determined no influence no influence reduction by heparin Stabilityat +4° C. stable Unstable after 7 days 100% loss of ~75% activityretained activity after 7 days incubation at +37° C. stable Unstablerelatively stable after 7 days 100% loss of ~60% activity retainedactivity after 7 days incubation stability stable Unstable not reportednot tested at +37° C. in after 7 days 100% loss of the presence of ~100%activity activity after 1 the dog's retained day incubation serum

The asterisks in the tables mean information on activity determinationand biochemical characterization (*-*****).

The methods of activity determination and biochemical characterizationused depend of the concentration of manillase in the analyzed samples.Therefore, they were successively extended by the appropriate techniquesin the successive steps of purification.

* Activity determination-turbidity reduction test ** Activitydetermination-turbidity reduction test Protein content determination(E₂₈₀, Pierce BCA method) SDS-PAGE (SDS-Polyacrylamide GelElectrophoresis) Hemoglobin determination *** Activitydetermination-turbidity reduction test Protein content determination(E₂₈₀, Pierce BCA method) SDS-PAGE-Western Blot (anti human hemoglobinantibody) **** Activity determination-turbidity reduction test Proteincontent determination (E₂₈₀, Pierce BCA method) SDS-PAGE-Western Blotanti human hemoglobin antibody, SDS-PAGE-Western Blot anti Con Aantibody SDS-PAGE-Western Blot-anti peptide antibodies ***** MALDIProtein content determination (Pierce BCA method) SDS-PAGE-WesternBlot-anti peptide antibodies

Binding of manillase to Concanavalin A shows that this hyaluronidase isa glycoprotein, whose sugar components are terminated withα-D-mannopyranosyl or α-D-glucopyranosyl and sterically relatedresidues. Manillase-active samples showed two bands with almostidentical RF values in SDS-PAGE. Longer SDS-PAGE and different runningconditions were used for better separation of the bands. In theseexperiments two additional, weaker bands could be detected (FIG. 2). TheN-terminal part all of them (30 amino acids) was individually sequencedand showed again no difference in the N-terminus. Followingdeglycosylation with the endo-F-glycosidase (PNGase) it was observedthat all four bands resulted in a single band, with a reduction in MW ofabout 3.

Therefore, it is quite likely that the observed differences inelectrophoretic mobility are due to differences in the glycosylationpattern of manillase molecules. The neuramimidase, O-endo-glycosidaseand neuramimidase plus O-glycosidase treatments have no influence on themolecular weight of the purified enzyme (FIG. 3). These results haveshown that manillase contains at least one N-linked oligosaccharidechain. The O-linked carbohydrate chains could not be detected with themethod used.

As the concluding purification step, the RP-chromatography was carriedout. Although the enzymatic activity could not be detected any more, thesalts and peptide protease inhibitors could be removed (FIG. 4). Thefractions containing protein were characterized further with the help ofMALDI. The molecular weight of manillase determined with the aid ofMALDI was 58.3.

Heparin has no influence on the activity of this hyaluronidase (FIG. 5).Manillase is many fold more stabile than Hirudo medicinalishyaluronidase (FIG. 6). Moreover, the samples of partly purifiedmanillase showed very high stability in the dogs and rats plasma withinthe −20 to +37 range.

The preparation of HA-affinity matrices has been described in theliterature (Tengblad A., Biochim. Biophys. Acta, 1979, 578, 281–289).This HA-matrix was used for the purification of the cartilagehyaluronate binding proteins or proteoglycan protein-keratan sulfatecore (Christner J. E., Anal. Biochem., 1978, 90, 22–32) from the samesource. The HA-binding protein (HABP), purified with the aid of thisaffinity matrix, was used further in histochemical studies concerningthe distribution of the hyaluronate receptors (Green S. J. et al., J.Cell Science, 1988, 89, 145–156; Chan F. L. et al., J. Cell. Biol.,1997, 107, 289–301) or hyaluronan (Waldenström A. et al., 1991, J. Clin.Invest, 88,1622–1628; Waldenström A. et al., Eur. J. Clin. Invest, 1993,23, 277–282) in the tissues.

However, the method of the preparation of this gel developed in ourlaboratory enables one to produce gels of exactly defined concentrationof HA-fragments (1 to 15 mg/ml). This, in turn, enables one to use suchgels not only for purification of hyaluronan-binding proteins but alsofor their separation, by taking advantage of their different affinity tohyaluronan. This selective separation can be controlled by using ofHA-fragments of different length. Such separation will enable one tobetter characterization many receptors of biological relevance (e.g. inoncology).

HA-matrices prepared according to the method described can be appliedfor the:

-   1) purification of known HA-binding proteins-   2) purification of unknown HA-binding proteins-   3) identification of the new HA-binding proteins-   4) purification of hyaluronidases

HA-fragments obtained by the method described in the present inventioncan be characterized with the use of modern analytical methods (NMR,MALDI-MS) and applied in the research on protein—protein interactions.Furthermore, these fragments can be used in the research concerningangiogenesis and neovascularization processes

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE—CBB staining)of the protein standard, manillase sample (after Diol-LiChrospherchromatography).

-   -   1—wide range protein standard    -   2—Manillase, 4 μg    -   3—Orgelase, 6 μg    -   4—Hemoglobin, 40 μg

FIG. 2: a) SDS-PAGE (CBB staining) and

-   -   b) SDS-PAGE—Western blot of four manillase-active samples (lines        3–6) after HA —affinity chromatography. Rabbit P3-2A polyclonal        anti-peptide antibody was used in this experiment.

FIG. 3: SDS-PAGE (CBB) of the following samples:

-   -   1—LW-MM—low weight molecular marker (BioRad)    -   2—Manillase    -   3—N-Glycosidase F (PNGase F)    -   4—Manillase after treatment with PNGase F    -   5—Manillase after treatment with O-glycosidase    -   6—Manillase after treatment with O-glycosidase and neuramimidase    -   7-O-glycosidase and neuramimidase    -   8—molecular weight marker (MWM—prestained BioRad)

FIG. 4: Reverse-Phase-Chromatography of

-   -   a) Ribonuclease standard    -   b) manillase sample (specific activity 140 kU/mg)

FIG. 5: Influence of heparin on hyaluronidase activity of manillase(-◯-) and bovine testes hyaluronidase (-●-) X-axis: IU heparin; Y-axis:% activity left

FIG. 6: Stability measurement of hyaluronidases in buffer and plasma:

-   -   (a) manillase (4° C.), (b) manillase (−20° C.)    -   (c) manillase (37° C.),    -   (d) bovine testes hyaluronidase (Y) and Hirudo medicinalis        hyaluronidase (A)    -   X-axis: days of incubation; Y-axis: WHO (IU) units

FIG. 7: Amino acid sequence of native manillase obtained by sequencingof the isolated and purified protein from Hirudinaria manillensisaccordning the invention (corresponds to SEQ ID No. 1)

FIG. 8: Nuclectide (upper lines) and amino acid sequence of arecombinant manillase clone (clone 21); (corresponds to SEQ ID. Nos. 2,3)

FIG. 9: Nucleotide (upper lines) and amino acid sequence of arecombinant manillase clone (clone 31); (corresponds to SEQ ID. Nos. 4,5)

FIG. 10: Nucleotide (upper tines) and amino acid sequence of arecombinant manillase clone (clone 31); (corresponds to SEQ ID. Nos. 6,7)

FIG. 11: E. coli expression vector for manillase

FIG. 12: Baculo donor plasmid for manillase

FIG. 13: Yeast expression vector for manillase

The invention is described in detail by the following examples. However,these examples do not limit the invention to the general materials,methods, physical parameters, compounds, biological materials,expression vectors and hosts etc. used in the experiments and indicatedin the examples. If not otherwise mentioned standard techniques wellknown in the prior art and generally available material were used.

EXAMPLE 1 General Remarks

A number of preliminary experiments were carried out using crudeextracts of Hirudinaria manillensis in order to establish thepurification procedure.

The following methods were chosen and verified: ammonium sulfateprecipitation procedure, cation and anion exchange chromatography,affinity chromatography with the aid of Heparin-Fractogel, ConA-Sepharose, Hydrophobic Interaction Chromatography (HIC) onOctyl-Sepharose, Propyl- Phenyl-, Butyl-Fractogel, preparativeisoelectric focusing and preparative electrophoresis.

The results show that acid and ammonium precipitation, cation exchange,Con A-Sepharose, Propyl-Fractogel HIC and Diol-LiChrospher andHyaluronic acid fragments-Sepharose (HA-Sepharose) chromatography aresuitable for the purification of the manillase. The HA-Sepharose matrixprepared in our laboratory was successfully used for the purification ofthis glycosidase.

All preparations were carried out in the cold unless otherwisementioned.

The purification was done according to the scheme shown above (Tab. 1).

EXAMPLE 2 Preparation of the Starting Material for the Purification;Preparation of Leech Heads.

Hirudinaria manillensis leeches collected in Bangladesh were immediatelyshock-frozen and then stored at −40° to −80°. They were decapitated infrozen state, the weight of the heads amounting to ca. 5% of the body.

EXAMPLE 3 Extraction Procedure of Manillase from Leech Heads

In a representative purification, 1 kg of frozen leech heads werehomogenized in a Waring Blender with 2500 ml of cold 0.1 M acetic acidbuffer pH 4.0 containing 0,025% thimerosal and 17 mg/ml of trehalose(Merck KGaA, Art. No. 1.08216). The homogenate was stirred gently andthe following protease inhibitors were added immediately:

1. PMSF 1.7 mg/ml 10.0 mM 2.Leupeptin 10.0 μg/ml 20.0 μM 3.Pepstatin A0.7 μg/ml 1 μM 4. EGTA 380.35 μg/ml 1.0 mM 5.p-APMSF 40.0 μg/ml 20.0 μM

Stirring was continued for 4 hour in the cold and centrifuged at 4900rpm for 20 minutes. The supernatant solution (supernatant I) wascollected and pooled with supernatant II subsequently obtained byextracting the tissues pellet.

The pooled supernatants represent Stage I material.

The procedure is summarized in the following scheme:

*Activity determination and biochemical characterization of the sampleswas performed with the aid of activity determination—turbidity reductiontest and protein content determination (E₂₈₀, Pierce BCA method,SDS-PAGE). It was impossible to measure the enzyme activity in the leechhomogenate, because of the very high content of hemoglobins (measuredwith the hemoglobin determination kit, Merck KGaA, 13851) and otherproteins. Moreover, the hyaluronidase activity could not be measured inthe stage prior to the acid precipitation. The final specific activities(activity per mg of protein) of these extracts were about 10–30 WHOUnits. According to SDS-PAGE, the crude extracts contained large amountsof different proteins, the major ones having a molecular weight of ˜120,55–60, 45, 31, 28, 22, 15 and 14-10.

EXAMPLE 4 Ammonium Sulfate Precipitation Procedure of the Stage IMaterial

Next, the ammonium sulfate precipitation procedure was chosen as thefirst step of the purification of manillase and resulted in a ˜5-fold ofenrichment of this enzyme.

Enzymatically inert material was precipitated from Stage I crude extractby adding slowly solid ammonium sulfate (Merck KGaA) to 36% w/v at +4°C. This mixture was stirred for 1 hour and centrifuged. The precipitatewas discarded. The supernatant was dialyzed against running de-ionizedwater overnight, and 24 hours against 20 mM phosphate buffer pH 6.0. Thefinal specific activities of these extracts were about 40–150 WHO Units.According to SDS-PAGE, the stage II extracts contain large amounts ofdifferent proteins.

EXAMPLE 5 Cation Exchange Chromatography

The cation exchanger was used in a batch adsorption mode. An enzyme richdialyzed sample (stage II) was incubated overnight with 1 l FractogelEMD SO₃ ⁻650 (S) cation exchanger, Merck KGaA, Art. No. 16882. After theincubation was finished by centrifugation, the cation exchanger waswashed with the buffer, centrifugated again and HPLC-Superformace columnwas filled with the gel. After washing the column with 20 mM phosphatebuffer pH 4.9 the bound proteins were eluted from the column with thesame sodium phosphate buffer pH 6.0 containing a linear 0 to 1 Mgradient of NaCl. Fractions were collected every 3 min (9 ml) and theabsorbance at 280 nm was monitored. Manillase was eluted at 0.15 to 0.18M NaCl concentrations. The activities and protein contents of allfractions were measured and the fractions were pooled and dialyzedovernight against 20 mM phosphate buffer pH 6.0 containing sodium azideand 17 mg/ml trehalose.

Determination of the concentration of proteins, specific activities ofthe “pools”, and SDS-PAGE analysis were carried out. In spite of verygood yields (activity) and high specific activity (WHO activity unitsper mg of protein, corresponds to IU), a mixture of many proteins wasstill shown by the results of SDS-PAGE analysis of the samples. Thecation exchange chromatography with the aid of Fractogel EMD SO₃ ⁻650(S)® (Merck KGaA, Germany) resulted in a very high purification factorof ˜10 to 50. This step is very effective in reducing hemoglobinimpurities. Moreover, we have found that the batch procedure was a veryuseful initial step for handling large volumes of stage II supernatant(5–16 l).

EXAMPLE 6 Concanavalin A-Sepharose Affinity Chromatography

The further purification of the enzyme-rich pools after cation exchangerwas done with the aid of Con A lectin affinity chromatography.Commercially available Con A-Sepharose®) from Pharmacia Biotech, Art.17-0440-01, was washed with an acetic buffer 0.1 M+0.5 M NaCl pH 8.0;0.1 M boric acid+0.1% Triton X 100 pH 6.0 and finally with 0.1 M aceticbuffer+0.5 M NaCl pH 6.0. The sample was dialyzed overnight against 20mM acetic buffer+0.5 mM NaCl+1 mM CaCl₂+1 mM MgCL₂ pH 6.0+1 mM MnCl₂,applied at room temperature to a 1000 ml Con A column and eluted 2 hwith the 510 ml of 100 mM acetic acid buffer+0.5 M NaCl +1 mM CaCl₂+1 mMMgCL₂ pH 6.0+1 mM MnCl₂.

This was followed by desorption with the aid of the same buffercontaining 0.5 M methyl-α-D-mannopyranoside. The elution wascontinuously monitored at 280 nm. The 3 ml fractions that had beencollected were assayed for hyaluronidase activity. The active fractionswere pooled and dialyzed overnight against 20 mM phosphate buffer pH 6.0containing sodium azide and 17 mg/ml trehalose. Determination of theconcentration of proteins, specific activities of the “pools”, andSDS-PAGE analysis was carried out. This step was very effective inremoving the rest of hemoglobin. The Con A chromatography resulted in a4–10 purification factor. This factor differed, depending on the qualityof the starting material.

EXAMPLE 7 Propyl Fractogel Hydrophobic Interaction Chromatography

To hyaluronidase active Con A-pools ammonium sulfate were added to afinal concentration of 2 M. The samples were then incubated 1 h at roomtemperature with 150 ml Propyl-Fractogel EMD Propyl 650 (S)®, MerckKgaA, Germany, Art. No. 1.10085, equilibrated with 0.1 M phosphatebuffer pH 7.0, containing 2 M ammonium sulfate. After the incubation wasfinished the gel was washed twice with the same buffer, and theHPLC-Superformance (2.6 cm×60 cm) column was prepared. The boundproteins were eluted with 0.1 M phosphate buffer pH 7.0. The 6 mlfractions were collected every 3 min, directly dialyzed againstde-ionized water (2–3 h) and, then against 20 mM phosphate buffer pH6.0. The fractions were assayed for hyaluronidase activity. The activefractions were pooled and dialyzed overnight against 20 mM phosphatebuffer pH 6.0 containing sodium azide and 17 mg/ml trehalose. Theprotein and activity determination of the pools was carried out.

The purification factor at this chromatography step was about 3 to 5. Asmall amount of Con A released from the carrier gel in the previous stepwas removed together with other protein impurities.

EXAMPLE 8 Preparation of Hyaluronic Acid Oligosaccharide Affinity Column

(a) Hydrolysis of Hyaluronan (HA) with Bovine Testes Hyaluronidase

Hyaluronic acid, 7 g was dissolved in 1.25 I of 0.1 M sodium acetatebuffer containing 0.15 NaCl and 0.5 mM EDTA, pH 5.2 by mixing overnightat 4° C. in the presence of toluene. Thereafter pH of HA containingsolution was adjusted to 5.2 and after warming up to 37° C., bovinetestes hyaluronidase (Merck KGaA; 700 WHO units/mg) was added. For 7 gof HA, 210 mg of enzyme dissolved immediately before use in 50 ml of theabove buffer were used. Hydrolysis was allowed to proceed for 30 min at37° C. with constant stirring, and terminated by heating for 5 min at100° C. in a boiling water bath. The reaction mixture was clarifiedthrough centrifugation for 30 min at 10 000 g, denatured proteincontaining sediment was discarded and supernatant filtered through 0.2μm filter, on which a glass fiber prefilter was placed. Clarifiedsolution containing HA oligosccharides (HAOS) was fractionated byfiltration through tree Diaflo ultrafiltration membrane (Amicon) withdifferent molecular cut off values as follows.

(b) Fractionation of HAOS by Ultrafiltration

HAOS—containing solution from the previous step was filtered through 30YM Diaflo ultrafiltration membrane. Retentate was saved for otherstudies while filtrate was subjected to the second ultrafiltrationthrough 10 YM Diaflo ultrafiltration membrane. Again, retentate wassaved for other studies while the solution passing through 10 YM wassubjected to the last ultrafiltration through 3 YM Diaflo membrane.Thereafter, retentate containing HA-OS, about 10 ml of the solution, wasused for further purification. This fraction: HAOS 3–10 was purified asfollows and further used for coupling to Sepharose.

(c) Purification of HAOS 3–10

HA-OS 3–10 were purified (desalted) on Biogel P2® column. This column (4cm×100 cm) was packed with Biogel 2 medium®, 200–400 mesh (BioRad), andwashed with 5 column volumes of water (Milli Q, Millipore). HAOS 3–10fraction obtained from the previous step (15 ml; 1.5 g ofoligosaccharides) was applied to this column. The column was eluted withwater; 15 ml fraction were collected and analyzed for the presence of HAoligosaccharides. Oligosaccharide containing fractions eluted beforesalts (the latter detected with AgNO3) were combined and concentratedagain on 3 YM Diaflo membrane.

(d) Analysis of HAOS 3–10

To determine the coupling efficiency of the Sepharose, gel (the samebatch) was washed and suspended in water as to prepare a 50% slurry.From the suspension of Sepharose-HAOS 3–10 conjugate and Sepharose usedas a control, 100 μl aliquots were withdrawn in triplicate and added to2.5 ml of 2.2 N trifluoroacetic acid (TFA, Merck KgaA) in teflon screwcapped tube. For hydrolysis, the mixture were flushed with argon andincubated at 100° C. for 16 h. At the end of hydrolysis, samples weredried under nitrogen, re-suspended in water and used for thedetermination of glucosamine and uronic acid. To determine the extent ofuronic acid and glucosamine decomposition for each of the hydrolysis,control samples containing known amounts of UA or GlcNAc were included,and incubated under the same conditions.

Under conditions described above 5, 8, 9, 11 and 15 mg of HAOS 3–10 werecoupled per 1 ml of drained Sepharose gel in two independentexperiments. This results are based on the UA and glucosamine assays.

(e) Assay Used

The content of the uronic acid in the samples analyzed was determinedaccording to Bitter T. and Muir H. M., Anal. Biochem., 1962, 4, 330–334.

The hexosamine amounts were analyzed with the method of Rondle C. J. M.and Morgan W. T. J., Biochem. J., 1955, 61, 586–593.

EXAMPLE 9 Hyaluronic Aci-d Fragments Sepharose Chromatography(HA-Sepharose Chromatography)

The chromatography matrices containing 8 to 10 mg/ml were prepared asindicated. The enzyme containing sample was dialyzed against 20 mMacetic buffer+0.15 M NaCl pH 4.0 and applied to the 25 ml HA-Sepharosecolumn. After washing with the same buffer, the elution was done withthe 20 mM acetic buffer with a 0.15 to 1 M gradient of NaCl.

The 1 ml fractions were tested in the hyaluronidase-activitydetermination test, pooled, dialyzed overnight against 20 mM phosphatebuffer pH 6.0 containing sodium azide and 17 mg/ml trehalose. Theprotein and activity determination of the pools was carried out. Thepurification factor of this chromatography step was about 3.

EXAMPLE 10 Diol-LiChrospher Chromatography

A 20 ml active sample dialyzed against Milli-Q-H₂O was applied on theDiol-LiChrospher column. The column was then equilibrated with 15 mlMilli-Q-H₂O and washed 5 min with 2 ml water. The elution of the activesample was done 15 min with 20 mM acetic buffer pH 5.9 (gradient, 0 to 5mM NaCl) and 35 min with gradient 20 mM to 100 mM acetic acid buffer pH5.5 containing 5 mM NaCl. The fractions were assayed for hyaluronidaseactivity. The active fractions were pooled and dialyzed overnightagainst 20 mM phosphate buffer pH 6.0 containing sodium azide and 17mg/ml trehalose. The protein and activity determination of the pools wascarried out. The purification factor: 3.

EXAMPLE 11 RP 18E Chromatography

This purification step can be used only as the last one and is aimed toobtain the sample devoid of salts and other protein impurities (e.g.peptide protease inhibitors). The hyaluronidase activity was completelylost, because manillase is not resistance to organic solvents used inthis step. Manillase sample was applied to the RP 18e column. The 0.25ml/min fractions were collected. The elution was done in the presence of0.1% TFA and, gradient water to 99% of acetonitrile was used. TheRP-purified samples can be used directly for amino acid sequencing,MALDI measurement, carbohydrate structure analysis and as standard forpurification of other batches of manillase.

EXAMPLE 12 Activity Determination—Turbidity Reduction Test

The hyaluronidase activity determination was done with the turbidityreduction measurements. Commercially available preparations ofhyaluronan (isolated from the different animal tissues and fluids, e.g.human cord, rooster comb) and hyaluronidases (endo-β-glucosaminidasesfrom bovine testes, porcine testes, bee venom; lyases from Streptomyceshyalurolyticus) were used for establishing suitable activity assayconditions. The endo-β-glucuronidase from Hirudo medicinalis waspartially purified in our laboratory.

Hyaluronan stock solution (conc. 2 mg/ml) was prepared by dissolving HAin 0.3 M phosphate buffer pH 5.3. This solution was diluted with thesame buffer to a concentration of 0.2 mg/ml directly before the test.The enzyme-containing samples were diluted to an appropriate amount ofenzyme (0.5–5 WHO units) with 20 mM phosphate buffer containing 0.01% ofbovine albumin and 77 mM of NaCl (enzyme dilution buffer). To 0.1 ml ofthese samples, 0.1 ml hyaluronan (0.2 mg/ml) solution was added, mixedand incubated 45 minutes at 37° C. The test was done in duplicate. Thereaction was stopped by dilution with 1.0 ml of albumin reagent (0.1% ofalbumin dissolved in 80 mM acetic acid/40 mM sodium acetate buffer, pH3.75). After 10 min incubation at RT or 37° C. the optical density at600 nm was read and the activity was expressed in WHO (IU) units bycomparison (SLT-program) with a standard. The WHO preparation of bovinetesticular hyaluronidase (Humphrey J. H., Bull. World Health Org. 1957,16, 291–294) was used as standard.

EXAMPLE 13 Protein Estimation

The protein content of column eluents was determined by measuring theultraviolet absorbance of solutions at 280 nm. The protein concentrationof the pooled fractions was determined with the aid of Piercemicromethod. The BSA solution was used as a reference protein.

EXAMPLE 14 SDS-Page Electrophoresis

Electrophoresis was done according to Laemmli procedure (Nature, 1970,227, 680–685). The following gels were used: 4 to 20% gradient or 12.5%separating gels with 4% stacking gel. Samples were subjected toelectrophoresis in the presence of sodium dodecyl sulfate andβ-mercaptoethanol. Proteins were visualized after staining withCoomassie brilliant blue and/or Silver staining (according to Pharmaciainstruction).

EXAMPLE 15 Isoelectric Focusing

To pursue isoelectric focusing studies on the manillase preparation, theprotocol provided by supplier (Pharmacia) was adopted. Followingfocusing, the gel was fixed and silver stained (according to Pharmaciaprotocol).

EXAMPLE 16 Preparation of Immunoglobin from Immune Sera of Rabbits

(Anti-ConA, Anti-Hemoglobin and Anti-Peptide Rabbit Antibodies)

The rabbit sera were raised with the use of the following immunogens:concanavalin A lectin, mixture of hemoglobins and peptide-KLHconjugates. The peptide sequence was identical with that of the 14 aminoacid N-terminal part of manillase (KEIAVTIDDKNVIA) (SEQ ID NO: 16). Thesera were purified on the Protein A Sepharose (Pharmacia, 17-0780-01)column according to the standard Pharmacia instruction. The purity ofthe IgG samples were checked with the aid of SDS-PAGE and ELISA-test.

EXAMPLE 17 Western-Immunoblot Assay

Suitable aliquots of the samples and pre-stained protein marker of knownmolecular weight were subjected to SDS-PAGE as described above. Apre-stained BioRad molecular weight marker was used. The protein wastransferred electrophoretically from polyacrylamide gels (0.8 mA/cm2) toimmobile polyvinyldifluoride (PVDF) membranes in the presence oftransfer buffer for 100 min. The PVDF membrane was incubated withblocking solution (PBS, pH 7.5+2% fat free milk) for 1 h at roomtemperature. Next, the membrane was incubated 2 h at room temperaturewith the antibody, appropriately diluted with the blocking solution. Themembrane was washed with TBS+0.05% Tween 20, pH 7.5, and incubated for 2h at room temperature with (a second antibody) goat anti-rabbit-alkalinephosphate conjugate, BioRad. The membrane was washed two times withTBS+Tween 20 and incubated 10 min with BCIP alkaline phosphatasesubstrate solution. Adding a stopping buffer terminated the reaction.

EXAMPLE 18 Amino Acid Sequencing

The sequence of N-terminal 33 amino acid residues of the manillase wasobtained by Edman degradation. After SDS-PAGE of manillase-activesamples, the bands were transferred onto PDVF membrane, stained withCoomassie Blue, cut-out and sequenced. The same amino sequence was foundfor the sample obtained after the last purification step with the aid ofRP-column chromatography.

EXAMPLE 19 pH Dependence of Enzyme Activity

(For Hyaluronidase Isolated from Hirudinaria manillensis and Hirudomedicinalis Leech Heads)

Samples of hyaluronidase used in this experiment were extracted eitherfrom Hirudinaria manillensis or Hirudo medicinalis leech heads andpartially purified with the aid of ammonium sulfate precipitation andcation exchange chromatography. Each sample containing 500 WHO units/mlwas incubated at −20° C., +4° C. and 37° C. at a range of pHs from 2.6to 9.0 (20 mM acetic for pH 2.6 to 5; 20 mM phosphate buffer for pH 5 to9). The enzyme activity was measured after 1, 2 and 7 days incubationperiods. At both acid and alkaline extremes of pH, inhibition ofactivity to the same extent was observed for both hyaluronidases.However, during longer incubation periods manillase was more stable thenHirudo medicinalis hyaluronidase: e.g. after 7 days incubation at pH 7.0at +4° C. and 37° C.—manillase retained 75% and 60% of the startingactivity, respectively. The Hirudo medicinalis hyaluronidase incubatedat the same conditions was already inactive after 1 day.

EXAMPLE 20 Stability Measurement of Hyaluronidases in the Presence ofDog'S Serum (for Hyaluronidase Isolated from Hirudinaria manillensis andHirudo medicinalis Leech Heads)

The 5 kU/ml samples of manillase, Hirudo medicinalis and bovine testeshyaluronidase were diluted with dog's or rat's citrated plasma to afinal concentration of 250 U/ml. Next, these solutions were incubated at−20° C., +4° C. and +37° C. for 0 to 7 days. The controls containing thesame hyaluronidases, diluted in buffer were included in this experiment.Finally, the hyaluronidase activity was measured.

EXAMPLE 21 Contaminating Enzyme Activities

At each stage of the purification procedure for leech hyaluronidase, thepreparation was checked for other enzymes capable of degrading proteinwith the aid of universal protease substrate (Boehringer Mannheim, cat.no. 1080 733) according to Twining S. S. (Anal. Biochem., 1984, 143,30–34).

EXAMPLE 22 Influence of Heparin on Hyaluronidase Activity

Cleavage of a hyaluronan by hyaluronidases results in the liberation ofreducing sugars. The amount of the liberated sugars was measuredcolorimetrically by the modified method of Park (Park J. & Johnson M.;J. Biol. Chem. 1949, 181, 149). For the measurement of the influence ofheparin on the activity of manillase and bovine testes hyaluronidase,two activity determination were carried out: one in the presence ofheparin, and second without heparin. Hyaluronidase samples, 25 μl (3.2WHO units) were incubated 30 min at 37° C. with 25 μl of the heparin(Liquernin, Fa. Hoffmann LaRoche) solution, containing 0 to 24 units ofheparin. Then, 50 μl of hyaluronan (2.5 mg/ml) was added and theincubation was continued for 30 min at 37° C. The reaction wasterminated by heating for 2 min at 100° C. Next, 100 μl ofcarbonate-cyanide solution and 100 μl of potassium ferricyanide solutionwere added to the inactivated digest. The samples were heated in aboiling water bath for 15 min and then cooled in an ice bath.Afterwards, 0.75 μl of ferric ammonium sulfate solution was added to thereaction mixtures. After 15 min incubation at RT, the color developedwas measured at 690 nm in a Shimadzu spectrophotometer. Suitable blanksand no-enzyme controls were included in each assay. The expectedreducing sugar (glucuronic acid or N-acetyl-glucosamine, 1 to 15 μg) forthe type of sample under analysis was used as standard.

EXAMPLE 23 Deglycosylation of the Manillase

The samples of manillase were deglycosylated with the aid of PNGase Fenzyme (BioLabs Art. No. 701 L) according to supplier instruction. Thede-glycosylation was done under denaturing and native conditions. TheO-glycanase, neuramimidase and neuramimidase+O-glycanase treatments weredone according to Boehringer Mannheim standard prescriptions. Allsamples were characterized with the SDS-PAGE and activity determinationtest.

EXAMPLE 24 Construction of the E. coli Expression Vector (FIG. 11)

For expression in E. coli we used a modified version of the plasmidpASK75, which carries the tet promoter region. {Skerra, Gene 151,(1994), pp 131–135}. The modification we made by cloning a new linkerbetween the XbaI an Hind III sites. The new linker contains the ompAleader sequence, another multiple cloning site and a 6×His-tag (SEQ IDNO: 17) instead of the strep-tag.

Linker sequence which was cloned in pASK75 (SEQ ID NOS 18–20).

     XbaI 119  CTAGATAACG AGGGCAAAAA ATGAAAAAGA CAGCTATCGC GATTGCAGTGGCACTGGCTG          TATTGC TCCCGTTTTT TACTTTTTCT GTCGATAGCG CTAACGTCACCGTGACCGAC                         1

 MetLysLysT hrAlaIleAl aIleAlaVal AlaLeuAlaG                              ClaI    EcoRI   SstI    KpnI SmaI   BamHI179  GTTTCGCTAC CGTAGCGCAG GC AT CGA TGA ATT CGA GCT CGG TAC CCG GGG     CAAAGCGATG GCATCGCGTC CG TA GCT ACT TAA GCT CGA GCC ATG GGC CCC  14

 lyPheAlaTh rValAlaGln Al a          XhoI    SalI    PstI       Eco47III 230  ATC CCT CGA GGT CGACCT GCA GGC AGC GCTATGAGAGGATCGCATCACCATCACCA      TAG GGA GCT CCA GCTGGA CGT CCG TCG CGATACTCTCCTAGCGTAGTGGTAGTGGT                                      1

 AlaMetArgGlySerHisHisHisHisHi               Hind III 286  TCACTAATAGA     AGTGATTATCTTCGA  10

 sHis . . .

To construct the expression vector for manillase it was necessary tointroduce 5′ ′Cla I and 3′ Eco47III restriction sites by PCR method.Therefore the two primers 5′ ATC GAT AAA GAG ATT GCC GTG AC (SEQ ID NO:8) and 3′ GTT GTT TCC GAT GCT AAA GCG CT (SEQ ID NO: 9) were used. ThePCR product first was cloned into the PCR II vector system (Invitrogen)and sequenced.

After expressing and proving the activity of this recombinant manillasein a second PCR reaction the His-tag was removed and the start codon ofthe manillase gene was directly fused to the omp A leader sequence. Theprimers for this PCR reaction were:

5′ ACC GTA GCG CAG GCC AAA GAG ATT GCC GTG (SEQ ID NO: 10) and 3′ CACGGC AAT CTC TTT GGC CTG CGC TAC GGT (SEQ ID NO: 11).

In a second step the manillase gene was cloned into the modified pASK75vector using the rectrictionsites 5′ClaI and 3′ Eco47111.

EXAMPLE 25 Construction of the Baculo Donor Plasmid (FIG. 12)

For expression of manillase in the Baculo virus expression system theBac-To-Bac™ Baculovirus Expression System from Gibco Life Technologieswas used. To get a section system the Honeybee melitin leader sequencewas fused to the manillase gene and to introduce the restriction sites5′ BamHI and 3′ KpnI one single PCR reaction was carried out.

5′ Primer:

CGG ATC CAT GAA ATT CTT AGT CAA CGT TGC CCT TGT TTT TAT GGT CGT ATA CATTTC TTA CAT CTA TGC GAA AGA GAT TGC CGT GAC (SEQ ID NO: 12)3′ Primer:AAT GTT GAA GCA TAA GGT ACC (SEQ ID NO: 13)

The PCR product was cloned into the PCR II Vector (Invitrogen) andsequenced. Then the Melitin—Manillase Fusion was cloned into thepFastBac vector using the restriction sites 5′BamHI and 3′KpnI (FIG.12).

EXAMPLE 26 Construction of the Yeast Expression Vector (FIG. 13)

For expression in yeast we used the pichia multi copy expression system(Invitrogen). To construct the expression vector for manillase we usedthe PCR amplification method of the manillase gene in such a way thatcompatible restriction ends (5′ EcoR I, 3′Not I) are generated forligation into the appropriate vector (pPIC9K). Therefore the followingprimers were used:

5′ GTA GAA TTC AAA GAG ATT GCC GTG ACA (SEQ ID NO: 14) 3′ GAT GCT AATGTT GAA GCA TAA TGA GCG GCC GC (SEQ ID NO: 15)

Before transforming the Pichia Speroplasts the expression vector has tobe liniarized with Sal I.

EXAMPLE 26 Expression in E. COLI

In the expression vector pRG72, which contains the structural gene ofSarastatin fused to the ompA leader sequence, was transformed into W3110competent cells. The cells were grown to a mid-log phase, and thepromoter was then induced by adding 200 μg aTC/l. 1 h thereafter therecombinant manillase could be clearly detected.

EXAMPLE 27 Generation of Recombinant Baculoviruses and ManillaseExpression with the Bac-To-Bac Expression System

The donor plasmid pTD13 was transformed into DH10Bac competent cellswhich contain the bacmid with a mini-attTn7 target site and the helperplasmid. The mini-Tn7 element on the donor plasmid ca transpose to the amini-attTn7 target site on the bacmid in the presence of transpositionproteins provided by the helper plasmid. Colonies containing recombinantbacmids were identified by disruption of the lacZ gene. High molecularweight mini-prep DNA prepared from selected E. coli clones containingthe recombinant bacmid, and this DNA was then used to transfect insectcells.

Detailed description could be find in the instruction manual of theexpression kit.

EXAMPLE 28 Expression in Yeast

To be sure to have integrated the manillase gene the colonies have to bescreened for His⁺Mut⁺-mutants.

Using a single colony, inoculate 100 ml Medium i a 1 l flask. Growingconditions are: 28–30° C., 250 rpm, up to OD 2–6. To induce expression,first cetrifuge the culture, decant to supernatant and re-suspend thecell pellet in new medium using ⅕ of the original culture volume. Add100% methanol to a final concentration of 0.5% every 24 hours tomaintain induction. After max 6 days supernatant is analyzed by SDS-Pageand the activity assay.

1. A purified protein isolated from the leech species Hirudinariamanillensis having the biological activity of a hyaluronidase which isnot influenced in its activity by heparin, characterized in that it hasa molecular weight of 53–60 kD dependent on glycosylation.
 2. Aglycosylated protein according to claim 1 having a molecular weight of58 (±2) kD.
 3. A non-glycosylated protein according to claim 1 having amolecular weight of 54 (2) kD.
 4. A protein according to claim 1 havingan isoelectric point of 7.2–8.0.
 5. A protein according to claim 1having the amino acid sequence given in FIG. 7 (SEQ ID NO:1).
 6. Aprotein according to claim 1 having a specific enzymatic activityof >100 kU/mg protein.
 7. A protein of claim 1 having the amino acidsequence of SEQ ID NO:
 3. 8. A protein of claim 1 having the amino acidsequence of SEQ ID NO:
 5. 9. A protein of claim 1 having the amino acidsequence of SEQ ID NO:
 7. 10. A recombinant protein with the biologicalactivity of a hyaluronidase and a molecular weight of 55–59 kD dependenton glycosylation having an amino acid sequence with at least 94%homology to SEQ ID NO:3.
 11. A protein of claim 10, having at least 96%homology to SEQ ID NO:
 3. 12. A protein of claim 10, having at least 97%homology to SEQ ID NO:
 3. 13. A process for isolating and purifying theprotein of claim 1 comprising: (i) homogenization of heads of leeches ofthe species Hirudinaria manillensis with an acid buffer andcentrifugation, (ii) ammonium sulfate precipitation of the supernatantof step (i), (iii) cation exchange chromatography, (iv) concanavalin Aaffinity chromatography, (v) hydrophobic interaction chromatography,(vi) affinity chromatography on matrices coated with hyaluronic acidfragments, and (vii) gel permeation chromatography.
 14. A process ofclaim 13, further comprising: (viii) enzymatic or chemicalde-glycosylation of the purified protein.
 15. A protein having thebiological activity of a hyaluronidase which is not influenced in itsactivity by heparin and having a molecular weight of 53–60 kD dependenton glycosylation, obtainable by the process steps of claim
 13. 16. Aprotein according to claim 15 having a specific enzymatic activityof >100 kU/mg protein.
 17. A DNA sequence coding for a protein ofclaim
 1. 18. A DNA sequence coding for a protein of claim 15 comprisinga nucleotide sequence depicted in FIG. 8 (SEQ. ID NO: 2), FIG. 9 (SEQ.ID NO: 4) or FIG. 10 (SEQ ID NO: 6).
 19. A recombinant protein havingthe biological activity of a hyaluronidase encoded by any a DNA sequenceof claim
 18. 20. An expression vector comprising a DNA sequence of claim17.
 21. A host cell suitable for the expression of a protein of claim 19which was transformed with a vector comprising a DNA sequence for aprotein comprising a nucleotide sequence depicted in FIG. 8 (SEQ. ID NO:2), FIG. 9 (SEQ. ID NO: 4) or FIG. 10 (SEQ ID NO: 6).
 22. Apharmaceutical composition comprising a protein of claim 1 and apharmaceutically acceptable diluent, carrier, or excipient therefor. 23.A pharmaceutical composition comprising the protein of claim 7 and apharmaceutically acceptable diluent, carrier or excipient therefor. 24.A pharmaceutical composition according to claim 22, further comprisingheparin.
 25. A method of treating myocardial, cardiovascular andthrombotic disorders and tumors in a subject, comprising administering aprotein of claim 1 to said subject.